Pregnancy-specific glycoproteins: evolution, expression, functions, and disease associations.

Pregnancy-specific glycoproteins (PSGs) are members of the immunoglobulin superfamily and are closely related to the predominantly membrane-bound CEACAM proteins. PSGs are produced by placental trophoblasts and secreted into the maternal bloodstream at high levels where they may regulate maternal immune and vascular functions through receptor binding and modulation of cytokine and chemokine expression and activity. PSGs may have autocrine and paracrine functions in the placental bed, and PSGs can activate soluble and extracellular matrix bound TGF-β, with potentially diverse effects on multiple cell types. PSGs are also found at high levels in the maternal circulation, at least in human, where they may have endocrine functions. In a non-reproductive context, PSGs are expressed in the gastrointestinal tract and their deregulation may be associated with colorectal cancer and other diseases. Like many placental hormones, PSGs are encoded by multigene families and they have an unusual phylogenetic distribution, being found predominantly in species with hemochorial placentation, with the notable exception of the horse in which PSG-like proteins are expressed in the endometrial cups of the epitheliochorial placenta. The evolution and expansion of PSG gene families appears to be a highly active process, with significant changes in gene numbers and protein domain structures in different mammalian lineages, and reports of extensive copy number variation at the human locus. Against this apparent diversification, the available evidence indicates extensive conservation of PSG functions in multiple species. These observations are consistent with maternal-fetal conflict underpinning the evolution of PSGs.

Classification of Preeclamptic Placental Extracellular Vesicles Using Femtosecond Laser-fabricated Nanoplasmonic Sensors and Machine Learning

Placental extracellular vesicles (EVs) play an essential role in pregnancy by protecting and transporting diverse biomolecules that aid in fetomaternal communication. However, in preeclampsia, they have also been implicated in contributing to disease progression. Despite their potential clinical value, most current technologies cannot provide a rapid and effective means of differentiating between healthy and diseased placental EVs. To address this, we developed a fabrication process called laser-induced nanostructuring of SERS-active thin films (LINST), which produces nanoplasmonic substrates that provide exceptional Raman signal enhancement and allow the biochemical fingerprinting of EVs. After validating LINST performance with chemical standards, we used placental EVs from tissue explant cultures and demonstrated that preeclamptic and normotensive placental EVs have classifiably distinct Raman spectra following the application of both conventional and advanced machine learning algorithms. Given the abundance of placental EVs in maternal circulation, these findings will encourage immediate exploration of surface-enhanced Raman spectroscopy (SERS) as a promising method for preeclampsia liquid biopsies, while our novel fabrication process can provide a versatile and scalable substrate for many other SERS applications.

Circulating Placental Alkaline Phosphatase Expressing Exosomes in Maternal Blood Showed Temporal Regulation of Placental Genes

Background: Analysis of placental genes could unravel maternal-fetal complications. However, inaccessibility to placental tissue during early pregnancy has limited this effort. We tested if exosomes (Exo) released by human placenta in the maternal circulation harbor crucial placental genes.Methods: Placental alkaline phosphate positive exosomes (ExoPLAP) were enriched from maternal blood collected at the following gestational weeks; 6–8th (T1), 12–14th (T2), 20–24th (T3), and 28th−32nd (T4). Nanotracking analysis, electron microscopy, dynamic light scattering, and immunoblotting were used for characterization. We used microarray for transcriptome and quantitative PCR (qPCR) for gene analysis in ExoPLAP.Results: Physical characterization and presence of CD63 and CD9 proteins confirmed the successful ExoPLAP enrichment. Four of the selected 36 placental genes did not amplify in ExoPLAP, while 32 showed regulations (n = 3–8/time point). Most genes in ExoPLAP showed significantly lower expression at T2–T4, relative to T1 (p < 0.05), such as NOS3, TNFSF10, OR5H6, APOL3, and NEDD4L. In contrast, genes, such as ATF6, NEDD1, and IGF2, had significantly higher expression at T2–T4 relative to T1. Unbiased gene profiling by microarray also confirmed expression of above genes in ExoPLAP-transcriptome. In addition, repeated measure ANOVA showed a significant change in the ExoPLAP transcriptome from T2 to T4 (n = 5/time point).Conclusion: Placental alkaline phosphate positive exosomes transcriptome changed with gestational age advancement in healthy women. The transcriptome expressed crucial placental genes involved in early embryonic development, such as actin cytoskeleton organization, appropriate cell positioning, DNA replication, and B-cell regulation for protecting mammalian fetuses from rejection. Thus, ExoPLAP in maternal blood could be a promising source to study the placental genes regulation for non-invasive monitoring of placental health.

Identifying target sites for placental therapeutics through the comparison of normal term pregnancies and intrauterine growth restricted proteomes

A variety of pathologies, including intrauterine growth restriction (IUGR), have been linked to placental insufficiencies as important causal factors, however, little has been done to develop therapeutics that may improve placental development, structure and function. The placenta offers the opportunity to manipulate the in-utero environment without directly intervening with the fetus, accessible from the maternal circulation, a vital but temporary organ, the placenta is no longer required after birth. Developing therapeutics must involve multiple aspects of targeting and safety to ensure no off-target impact on the pregnant person or fetus as well as enhance efficiency of delivery. In addition to our studies on nanoparticle delivery to the placenta [1] we are developing targeting strategies to allow peripheral delivery via the maternal circulation. In this study we have performed the isolation of the microvillous membrane from human placental syncytiotrophoblast (the targeting cell) and via proteomic analysis identified potential targeting moieties, we have then compared this to publicly available data from pregnancies complicated by fetal growth restriction to ensure that we do not identify targets which would be reduced in FGR, resulting in less efficient targeting.

Longitudinal Changes In Maternal Circulating Micrornas As Potential Biomarkers of Specific Events During Healthy Pregnancy

Background: Novel high-resolution tools for pregnancy monitoring, including early detection of prenatal disorders, are needed. Changes in circulating microRNAs (c-miRNAs) during pregnancy could potentially inform about the functional status of the mother, the placenta and/or the fetus. However, whether c-miRNA profiles actually reflect distinct pregnancy-specific events at all stages remains unclear.Methods: Longitudinal large-scale RNAseq c-miRNA profiles at early, middle and late pregnancy, and after birth derived from eight women with healthy term pregnancies (n=32 plasma samples) were compared against corresponding circulating profiles derived from age-matched non-pregnant women (n=10). Data of fetal sex and growth indicators obtained during pregnancy evolution of the same women, were used to identify specific c-miRNA correlates in circulation.Results: 1449 c-miRNAs were detected in circulation in both pregnant and non-pregnant women with only 48 c-miRNAs differentially expressed relative to non-pregnant controls in at least one of the four studied stages (FDR<0.05). Surprisingly, c-miRNA subpopulations with reported prominent expression in various pregnancy-associated compartments (placenta, amniotic fluid, umbilical cord plasma and breast milk) were found collectively under-expressed in maternal circulation throughout pregnancy (p<0.05). Furthermore, we found a bias in global miRNAs expression in direct association with fetal sex right from the first trimester, in addition to a specific c-miRNA signature of fetal growth (R = 0.7, p < 0.01).Conclusion: Our results demonstrate the existence of temporal changes in c-miRNAs populations associated to distinct aspects of pregnancy, including correlates of placental function and lactation, as well as fetal gender and growth, revealing a wider potential of c-miRNAs as biomarkers of specific aspects of maternal health and fetal growth.

Methods of detection and isolation of trophoblast cells from trans-cervical specimens – a historical overview

Trophoblast cells can be detected and isolated from the cervical epithelial cells obtained via various techniques of trans-cervical samples collection such as a mucus aspiration, endocervical lavage, or standard cervical brushing in the early first trimester, even from the 5 weeks’ gestation. Isolated fetal cells can be used in the early prediction of fetal sex, prenatal diagnostics of the most common aneuploidies, and any other genetic abnormalities. Nevertheless, the collection of trophoblastic cells has limited efficacy compared to currently used methods of detection of free fetal DNA in maternal circulation or other protocols of invasive prenatal diagnostics available at later stages of pregnancy. In the past years, trans-cervical cell samples were collected mainly in women before planned pregnancy termination. The early trophoblastic cells isolation from women in ongoing pregnancies opens new perspectives for further studies focused on the elucidation of pathophysiology of numerous pregnancy-related complications.

Placental Exosomes Trigger Maternal Inflammation and Vascular Dysfunction in Preeclampsia

Preeclampsia is a pregnancy-specific disease associated with inadequate placental formation, chronic inflammation, and maternal vascular dysfunction. Preeclampsia affects about 5-8% of pregnant women and it is a prevalent cause of maternal mortality. The level and composition of exosomes in the maternal circulation are altered in preeclampsia, and studies have shown that the major source of this greater level of exosomes is the placenta. We propose that exosomal contents from the placenta trigger maternal inflammation and vascular dysfunction, thereby exacerbating the disease progression. This mini-review will focus on the content of placental exosomes and how they could contribute to the development of preeclampsia.

Isolation and Enrichment of Circulating Fetal Cells for NIPD: An Overview

Prenatal diagnosis plays a crucial role in clinical genetics. Non-invasive prenatal diagnosis using fetal cells circulating in maternal peripheral blood has become the goal of prenatal diagnosis, to obtain complete fetal genetic information and avoid risks to mother and fetus. The development of high-efficiency separation technologies is necessary to obtain the scarce fetal cells from the maternal circulation. Over the years, multiple approaches have been applied, including choice of the ideal cell targets, different cell recovering technologies, and refined cell isolation yield procedures. In order to provide a useful tool and to give insights about limitations and advantages of the technologies available today, we review the genetic research on the creation and validation of non-invasive prenatal diagnostic testing protocols based on the rare and labile circulating fetal cells during pregnancy.

SARS-CoV-2 and the Immune Response in Pregnancy with Delta Variant Considerations

As of September 2021, there has been a total of 123,633 confirmed cases of pregnant women with SARS-CoV-2 infection in the US according to the CDC, with maternal death being 2.85 times more likely, pre-eclampsia 1.33 times more likely, preterm birth 1.47 times more likely, still birth 2.84 times more likely, and NICU admission 4.89 times more likely when compared to pregnant women without COVID-19 infection. In our literature review, we have identified eight key changes in the immunological functioning of the pregnant body that may predispose the pregnant patient to both a greater susceptibility to SARS-CoV-2, as well as a more severe disease course. Factors that may impede immune clearance of SARS-CoV-2 include decreased levels of natural killer (NK) cells, Th1 CD4+ T cells, plasmacytoid dendritic cells (pDC), a decreased phagocytic index of neutrophil granulocytes and monocytes, as well as the immunomodulatory properties of progesterone, which is elevated in pregnancy. Factors that may exacerbate SARS-CoV-2 morbidity through hyperinflammatory states include increases in the complement system, which are linked to greater lung injury, as well as increases in TLR-1 and TLR-7, which are known to bind to the virus, leading to increased proinflammatory cytokines such as IL-6 and TNF-α, which are already elevated in normal pregnant physiology. Other considerations include an increase in angiotensin converting enzyme 2 (ACE2) in the maternal circulation, leading to increased viral binding on the host cell, as well as increased IL-6 and decreased regulatory T cells in pre-eclampsia. We also focus on how the Delta variant has had a concerning impact on SARS-CoV-2 cases in pregnancy, with an increased case volume and proportion of ICU admissions among the infected expecting mothers. We propose that the effects of the Delta variant are due to a combination of (1) the Delta variant itself being more transmissible, contagious, and efficient at infecting host cells, (2) initial evidence pointing to the Delta variant causing a significantly greater viral load that accumulates more rapidly in the respiratory system, (3) the pregnancy state being more susceptible to SARS-CoV-2 infection, as discussed in-depth, and (4) the lower rates of vaccination in pregnant women compared to the general population. In the face of continually evolving strains and the relatively low awareness of COVID-19 vaccination for pregnant women, it is imperative that we continue to push for global vaccine equity.